Nanoparticle contrast agents for diagnostic imaging

ABSTRACT

Compositions of nanoparticles functionalized with at least one zwitterionic moiety, methods for making a plurality of nanoparticles, and methods of their use as diagnostic agents are provided. The nanoparticles have characteristics that result in minimal retention of the particles in the body compared to other nanoparticles. The nanoparticle comprises a core, having a core surface essentially free of silica, and a shell attached to the core surface. The shell comprises at least one silane-functionalized zwitterionic moiety.

BACKGROUND

This application relates generally to contrast agents for diagnosticimaging, such as for use in X-ray/Computed Tomography (CT) or MagneticResonance Imaging (MRI). More particularly, the application relates tonanoparticle-based contrast agents, and methods for making and usingsuch agents.

Almost all clinically approved diagnostic contrast agents are smallmolecule based. Iodinated aromatic compounds have served as standardX-ray or CT contrast agents, while Gd-chelates are used for MagneticResonance Imaging. Although commonly used for diagnostic imaging, smallmolecule contrast agents may suffer from certain disadvantages such asleakage from blood vessel walls leading to short blood circulation time,lower sensitivity, high viscosity, and high osmolality. These compoundsgenerally have been associated with renal complications in some patientpopulations. This class of small molecule agents is known to clear fromthe body rapidly, limiting the time over which they can be used toeffectively image the vascular system as well as, in regards to otherindications, making it difficult to target these agents to diseasesites. Thus there is a need for a new class of contrast agents.

Nanoparticles are being widely studied for uses in medical applications,both diagnostic and therapeutic. While only a few nanoparticle-basedagents have been clinically approved for magnetic resonance imagingapplications and for drug delivery applications, hundreds of such agentsare still in development. There is substantial evidence thatnanoparticles have benefits over currently used small molecule-basedagents in terms of efficacy for diagnostics and therapeutics. However,the effect of particle size, structure, and surface properties on thein-vivo bio-distribution and clearance of nanoparticle agents is notwell understood. Nanoparticles, depending on their size, tend to stay inthe body for longer periods compared to small molecules. In the case ofcontrast agents, it is preferred to have maximum renal clearance of theagents from the body without causing short term or long term toxicity toany organs.

In view of the above, there is a need for nanoparticle-based contrastagents or imaging agents with improved properties, particularly relatedto renal clearance and toxicity effects.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a new class of nanoparticle-basedcontrast agents for X-ray, CT and MRI. The present inventors have foundthat nanoparticles functionalized with zwitterionic groups surprisinglyhave improved imaging characteristics compared to small moleculecontrast agents. The nanoparticles of the present invention havecharacteristics that result in minimal retention of the particles in thebody compared to other nanoparticles. These nanoparticles may provideimproved performance and benefit in one or more of the following areas:robust synthesis, reduced cost, image contrast enhancement, increasedblood half life, and decreased toxicity.

The present invention is directed to a composition comprising ananoparticle, its method of making and method of use.

One aspect of the invention relates to a composition comprising ananoparticle. The nanoparticle comprises a core, having a core surfaceessentially free of silica, and a shell attached to the core surface.The shell comprises at least one silane-functionalized zwitterionicmoiety. In one embodiment, the core comprises a transition metal. Inanother embodiment, the core comprises a transition metal compoundselected from the group consisting of oxides, carbides, sulfides,nitrides, phosphides, borides, halides, selenides, tellurides, orcombinations thereof. In one embodiment, the core comprises a metal withan atomic number ≧34.

In some embodiments, the composition comprises a nanoparticle comprisinga tantalum oxide core, having a core surface essentially free of silica,and a shell attached to the core surface, wherein the shell comprises atleast one silane-functionalized zwitterionic moiety. The nanoparticlehas an average particle size up to about 6 nm.

In some other embodiments, the composition comprises a nanoparticlecomprising a superparamagnetic iron oxide core, having a core surfaceessentially free of silica, and a shell attached to the core surface,wherein the shell comprises at least one silane-functionalizedzwitterionic moiety. The nanoparticle has an average particle size up toabout 50 nm.

In one or more embodiments, the invention relates to a diagnostic agentcomposition. The composition comprises a plurality of nanoparticles,wherein at least one nanoparticle of the plurality comprises a core,having a core surface essentially free of silica, and a shell attachedto the core surface. The shell comprises at least onesilane-functionalized zwitterionic moiety. In some embodiments, thecomposition further comprises a pharmaceutically acceptable carrier andoptionally one or more excipients.

One aspect of the invention relates to methods for making a plurality ofnanoparticles. The method comprises (a) providing a core, having a coresurface essentially free of silica, and (b) disposing a shell attachedto the core surface, wherein the shell comprises a silane-functionalizedzwitterionic moiety.

Another aspect of the invention is directed to a method comprisingadministering a diagnostic agent composition to a subject and imagingthe subject with an X-ray device. The diagnostic agent compositioncomprises a plurality of nanoparticles, wherein at least onenanoparticle of the plurality comprises a core and a shell. The shellcomprises at least one silane-functionalized zwitterionic moiety. In oneor more embodiments, the core comprises tantalum oxide.

In some embodiments, the method comprises administering a diagnosticagent composition to a subject, and imaging the subject with adiagnostic device. The diagnostic agent composition comprises aplurality of nanoparticles. At least one nanoparticle of the pluralitycomprises a core, having a core surface essentially free of silica, anda shell attached to the core surface. The shell comprises at least onesilane-functionalized zwitterionic moiety. In one or more embodiments,the method further comprises monitoring delivery of the diagnostic agentcomposition to the subject with the diagnostic device and diagnosing thesubject. In some embodiments, the diagnostic device employs an imagingmethod selected from the group consisting of magnetic resonance imaging,optical imaging, optical coherence tomography, X-ray, computedtomography, positron emission tomography, or combinations thereof.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 depicts a cross-sectional view of a nanoparticle comprising acore and a shell, in accordance with some embodiments of the presentinvention.

FIG. 2 describes organic acids and organic bases from which thezwitterionic functional groups may be formed.

FIGS. 3A, 3B, 3C and 3D describe silane-functionalized zwitterionicmoieties, which may react with the core to produce a shell comprisingsilane functional zwitterionic moieties.

DETAILED DESCRIPTION

The following detailed description is exemplary and is not intended tolimit the invention of the application or the uses of the invention.Furthermore, there is no intention to be limited by any theory presentedin the preceding background of the invention or the following detaileddescription.

In the following specification and the claims which follow, referencewill be made to a number of terms having the following meanings. Thesingular forms “a”, “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Approximating language, as usedherein throughout the specification and claims, may be applied to modifyany quantitative representation that could permissibly vary withoutresulting in a change in the basic function to which it is related.Accordingly, a value modified by a term such as “about” is not to belimited to the precise value specified. In some instances, theapproximating language may correspond to the precision of an instrumentfor measuring the value. Similarly, “free” may be used in combinationwith a term, and may include an insubstantial number, or trace amounts,while still being considered free of the modified term. For example,free of solvent or solvent-free, and like terms and phrases, may referto an instance in which a significant portion, some, or all of thesolvent has been removed from a solvated material.

One or more embodiments of the invention are related to a compositioncomprising a nanoparticle, as described in FIG. 1. The nanoparticle 10composition comprises a core 20, having a core surface 30 essentiallyfree of silica. In one or more embodiments, the core 20 contains atransition metal, for example, a compound of a transition metal element.The nanoparticle 10 further includes a shell 40, also referred to as acoating, attached to the core surface 30. The shell 40 comprises atleast one silane-functionalized zwitterionic moiety. Because the coresurface 30 is essentially free of silica, the silane-functionalizedzwitterionic moieties are not bound to silica, but are bound to the core20 at the core surface 30 without any intervening silica layer. Thesilane-functionalized zwitterionic moiety comprises a silane moiety anda zwitterionic moiety. As used herein, the term “zwitterionic moiety”refers to a moiety that is electrically neutral but carries formalpositive and negative charges on different atoms. Zwitterions are polarand usually have a high solubility in water and a poor solubility inmost organic solvents. In some embodiments, the “zwitterionic moiety”refers to a precursor to a zwitterionic moiety. In such embodiments, theprecursor undergoes a secondary or subsequent chemical reaction to forma zwitterionic moiety.

“Nanoparticle” as used herein refers to particles having a particle sizeon the nanometer scale, generally less than 1 micrometer. In oneembodiment, the nanoparticle has a particle size up to about 50 nm. Inanother embodiment, the nanoparticle has a particle size up to about 10nm. In another embodiment, the nanoparticle has a particle size up toabout 6 nm.

A plurality of nanoparticles may be characterized by one or more ofmedian particle size, average diameter or particle size, particle sizedistribution, average particle surface area, particle shape, or particlecross-sectional geometry. Furthermore, a plurality of nanoparticles mayhave a distribution of particle sizes that may be characterized by botha number-average size and a weight-average particle size. Thenumber-average particle size may be represented byS_(N)=Σ(s_(i)n_(i))/Σn_(i), where n_(i) is the number of particleshaving a particle size s_(i). The weight average particle size may berepresented by S_(W)=Σ(s_(i)n_(i) ²)/Σ(s_(i)n_(i)). When all particleshave the same size, S_(N) and S_(W) may be equal. In one embodiment,there may be a distribution of sizes, and S_(N) may be different fromS_(W). The ratio of the weight average to the number average may bedefined as the polydispersity index (S_(PDI)). In one embodiment,S_(PDI) may be equal to about 1. In other embodiments, respectively,S_(PDI) may be in a range of from about 1 to about 1.2, from about 1.2to about 1.4, from about 1.4 to about 1.6, or from about 1.6 to about2.0. In one embodiment, S_(PDI) may be in a range that is greater thanabout 2.0.

In one embodiment, a plurality of nanoparticles may have a particle sizedistribution selected from a group consisting of normal distribution,monomodal distribution, and bimodal distribution. Certain particle sizedistributions may be useful to provide certain benefits. A monomodaldistribution may refer to a distribution of particle sizes distributedabout a single mode. In another embodiment, populations of particleshaving two distinct sub-population size ranges (a bimodal distribution)may be included in the composition.

A nanoparticle may have a variety of shapes and cross-sectionalgeometries that may depend, in part, upon the process used to producethe particles. In one embodiment, a nanoparticle may have a shape thatis a sphere, a rod, a tube, a flake, a fiber, a plate, a wire, a cube,or a whisker. A nanoparticle may include particles having two or more ofthe aforementioned shapes. In one embodiment, a cross-sectional geometryof the particle may be one or more of circular, ellipsoidal, triangular,rectangular, or polygonal. In one embodiment, a nanoparticle may consistessentially of non-spherical particles. For example, such particles mayhave the form of ellipsoids, which may have all three principal axes ofdiffering lengths, or may be oblate or prelate ellipsoids of revolution.Non-spherical nanoparticles alternatively may be laminar in form,wherein laminar refers to particles in which the maximum dimension alongone axis is substantially less than the maximum dimension along each ofthe other two axes. Non-spherical nanoparticles may also have the shapeof frusta of pyramids or cones, or of elongated rods. In one embodiment,the nanoparticles may be irregular in shape. In one embodiment, aplurality of nanoparticles may consist essentially of sphericalnanoparticles.

A population of nanoparticles may have a high surface-to-volume ratio. Ananoparticle may be crystalline or amorphous. In one embodiment, asingle type (size, shape, and the like) of nanoparticle may be used, ormixtures of different types of nanoparticles may be used. If a mixtureof nanoparticles is used they may be homogeneously or non-homogeneouslydistributed in the composition.

In one embodiment, the nanoparticle may be stable towards aggregate oragglomerate formation. An aggregate may include more than onenanoparticle in physical contact with one another, while agglomeratesmay include more than one aggregate in physical contact with oneanother. In some embodiments, the nanoparticles may not be stronglyagglomerated and/or aggregated such that the particles may be relativelyeasily dispersed in the composition.

In one embodiment, the core comprises a transition metal. As usedherein, “transition metal” refers to elements from groups 3-12 of thePeriodic Table. In certain embodiments, the core comprises one or moretransition metal compounds, such as oxides, carbides, sulfides,nitrides, phosphides, borides, halides, selenides, and tellurides, thatcontain one or more of these transition metal elements. Accordingly, inthis description the term “metal” does not necessarily imply that azero-valent metal is present; instead, the use of this term signifiesthe presence of a metallic or nonmetallic material that contains atransition metal element as a constituent.

In some embodiments, the nanoparticle may comprise a single core. Insome other embodiments, the nanoparticle may comprise a plurality ofcores. In embodiments where the nanoparticle comprises plurality ofcores, the cores may be the same or different. In some embodiments, thenanoparticle composition comprises at least two cores. In otherembodiments, each of the nanoparticle composition comprises only onecore.

In some embodiments, the core comprises a single transition metalcompound. In another embodiment, the core comprises two or moretransition metal compounds. In embodiments where the core comprises twoor more transition metal compounds, the transition metal element or thetransition metal cation may be of the same element or of two or moredifferent elements. For example, in one embodiment, the core maycomprise a single metal compound, such as tantalum oxide or iron oxide.In another embodiment, the core may comprise two or more different metalelements, for example tantalum oxide and hafnium oxide or tantalum oxideand hafnium nitride, or oxides of iron and manganese. In anotherembodiment, the core may comprise two or more compounds of the samemetal element, for example tantalum oxide and tantalum sulfide.

In one embodiment, the core creates a contrast enhancement in X-ray orcomputed tomography (CT) imaging. A conventional CT scanner uses a broadspectrum of X-ray energy between about 10 keV and about 150 keV. Thoseskilled in the art will recognize that the amount of X-ray attenuationpassing through a particular material per unit length is expressed asthe linear attenuation coefficient. At an X-ray energy spectrum typicalin CT imaging, the attenuation of materials is dominated by thephotoelectric absorption effect and the Compton Scattering effect.Furthermore, the linear attenuation coefficient is well known to be afunction of the energy of the incident X-ray, the density of thematerial (related to molar concentration), and the atomic number (Z) ofthe material. For molecular compounds or mixtures of different atoms the‘effective atomic number,’ Z_(eff), can be calculated as a function ofthe atomic number of the constituent elements. The effective atomicnumber of a compound of known chemical formula is determined from therelationship:

$\begin{matrix}{Z_{eff} = \left\lbrack {\sum\limits_{k = 1}^{P}{w_{f_{k}}Z_{k}^{\beta}}} \right\rbrack^{1/\beta}} & \left( {{Eq}.\mspace{14mu} 1} \right)\end{matrix}$where Z_(k) is the atomic number of metal elements, P is the totalquantity of metal elements, and w_(f) _(k) is the weight fraction ofmetal elements with respect to the total molecular weight of themolecule (related to the molar concentration). The optimal choice of theincident X-ray energy for CT imaging is a function of the size of theobject to be imaged and is not expected to vary much from the nominalvalues. It is also well known that the linear attenuation coefficient ofthe contrast agent material is linearly dependent on the density of thematerial, i.e., the linear attenuation coefficient can be increased ifthe material density is increased or if the molar concentration of thecontrast material is increased. However, the practical aspects ofinjecting contrast agent material into patients, and the associatedtoxicity effects, limit the molar concentration that can be achieved.Therefore it is reasonable to separate potential contrast agentmaterials according to their effective atomic number. Based onsimulations of the CT contrast enhancement of typical materials for atypical CT energy spectrum with a molar concentration of approximately50 mM, it is estimated that materials with effective atomic numbergreater than or equal to 34 may yield appropriate contrast enhancementof about 30 Hounsfield units (HU), or 3% higher contrast than water.Therefore, in certain embodiments the core comprises material having aneffective atomic number greater than or equal to 34. See, e.g., Chapter1 in Handbook of Medical Imaging, Volume 1. Physics and Psychophysics,Eds. J. Beutel, H. L. Kundel, R. L. Van Metter, SPIE Press, 2000.

A core that contains transition metals with relatively high atomicnumber as described above may provide embodiments having certaindesirable characteristics. In such embodiments, the core issubstantially radiopaque, meaning that the core material prohibitssignificantly less X-ray radiation to pass through than materialstypically found in living organisms, thus potentially giving theparticles utility as contrast agents in X-ray imaging applications, suchas computed tomography (CT). Examples of transition metal elements thatmay provide this property include tungsten, tantalum, hafnium,zirconium, molybdenum, silver, and zinc. Tantalum oxide is oneparticular example of a suitable core composition for use in X-rayimaging applications. In one or more embodiments, the core of thenanoparticle comprises tantalum oxide and the nanoparticle has aparticle size up to about 6 nm. This embodiment may be particularlyattractive for applications in imaging techniques that apply X-rays togenerate imaging data, due to the high degree of radiopacity of thetantalum-containing core and the small size that aids rapid renalclearance, for example.

In some embodiments, the core of the nanoparticle comprises at leastabout 30% transition metal material by weight. In certain embodiments,the core comprises at least about 50% transition metal material byweight. In still further embodiments, the core comprises at least about75% transition metal material by weight. Having a high transition metalmaterial content in the core provides the nanoparticle with higherdegree of radiopacity per unit volume, thereby imparting more efficientperformance as an contrast agent.

In another embodiment, the core comprises material that exhibitsmagnetic behavior, including, for example, superparamagnetic behavior.The “superparamagnetic material” as used herein refers to material thatmay exhibit a behavior similar to paramagnetism even when attemperatures below the Curie or the Néel temperature. Examples ofpotential magnetic or superparamagnetic materials include materialscomprising one or more of iron, manganese, copper, cobalt, or nickel. Inone embodiment, the superparamagnetic material comprisessuperparamagnetic iron oxide. In some embodiments, the nanoparticles ofthe present invention may be used as magnetic resonance (MR) contrastagents. These nanoparticles may yield a T2*, T2, or T1 magneticresonance signal upon exposure to a magnetic field. In one or moreembodiments, the core of the nanoparticle comprises superparamagneticiron oxide and the nanoparticle has a particle size up to about 50 nm.

In one embodiment, the nanoparticle 10 comprises a shell 40substantially covering the core 20. This shell 40 may serve to stabilizethe core 20, i.e., the shell 40 may prevent one core 20 from contactingan adjacent core 20, thereby preventing a plurality of such nanoparticle10 from aggregating or agglomerating as described herein, or bypreventing leaching of metal or metal oxide, for instance, on the timescale of in-vivo imaging experiments. In one embodiment, the shell 40may be of a sufficient thickness to stabilize the core 20 and preventsuch contact. In one embodiment, the shell 40 has an average thicknessup to about 50 nm. In another embodiment, the shell 40 has an averagethickness up to about 3 nm.

As used herein, the term “substantially covering” means that apercentage surface coverage of the nanoparticle is greater than about20%. Percentage surface coverage refers to the ratio of nanoparticlesurface covered by the shell to the surface area not covered by theshell. In some embodiments, the percentage surface coverage of thenanoparticle may be greater than about 40%.

In some embodiments, the shell may facilitate improved water solubility,reduce aggregate formation, reduce agglomerate formation, preventoxidation of nanoparticles, maintain the uniformity of the core-shellentity, or provide biocompatibility for the nanoparticles. In anotherembodiment, the material or materials comprising the shell may furthercomprise other materials that are tailored for a particular application,such as, but not limited to, diagnostic applications. For instance, inone embodiment, the nanoparticle may further be functionalized with atargeting ligand. The targeting ligand may be a molecule or a structurethat provides targeting of the nanoparticle to a desired organ, tissueor cell. The targeting ligand may include, but is not limited to,proteins, peptides, antibodies, nucleic acids, sugar derivatives, orcombinations thereof. In some embodiments, the nanoparticle furthercomprises targeting agents such that, when used as contrast agents, theparticles can be targeted to specific diseased areas of the subject'sbody. In some embodiments, the nanoparticles may be used as blood poolagents.

The cores may be covered with one or more shells. In some embodiments, aplurality of cores may be covered with the same shell. In oneembodiment, a single shell may cover all the cores present in thenanoparticle composition. In some embodiments, the individual cores maybe covered with one or more shells. In another embodiment, all the corespresent in the nanoparticle may be covered with two or more shells. Anindividual shell may comprise the same material or may comprise two ormore different materials. In embodiments where the core may be coveredwith more than one shell, the shell may be of the same or of differentmaterial.

In one embodiment, the shell comprises at least onesilane-functionalized zwitterionic moiety, wherein thesilane-functionalized zwitterionic moiety comprises a silane moiety anda zwitterionic moiety. In some embodiments, the silane moiety of thesilane-functionalized zwitterionic shell is directly attached to thecore.

In one embodiment, the shell comprises a plurality of silane moieties,wherein at least one of the plurality of silane moieties isfunctionalized with at least one zwitterionic moiety. In someembodiments, the shell comprises silane-functionalized zwitterionicmoieties and silane-functionalized non-zwitterionic moieties. In suchembodiments, a ratio of silane-functionalized zwitterionic moieties tosilane-functionalized non-zwitterionic moieties is from about 0.01 toabout 100. In some other embodiments, the ratio of silane-functionalizedzwitterionic moieties to silane-functionalized non-zwitterionic moietiesis from about 0.1 to about 20.

In some embodiments, the shell comprises a plurality ofsilane-functionalized zwitterionic moieties. The term “plurality ofsilane-functionalized zwitterionic moieties” refers multiple instancesof one particular silane moiety, functionalized with at least onezwitterionic moiety. The silane moieties may be the same or different.In one embodiment, each core is surrounded by a plurality ofsilane-functionalized zwitterionic moieties, wherein all the silanemoieties are of the same type. In another embodiment, each core issurrounded by a plurality of silane-functionalized zwitterionicmoieties, wherein the silane moieties are of different types. In oneembodiment, each of the plurality of silane moieties is functionalizedwith at least one zwitterionic moiety. In one embodiment, at least oneof the plurality of silane moieties is functionalized with azwitterionic moiety such that each nanoparticle, on average, comprisesat least one zwitterionic moiety. In one or more embodiments, eachnanoparticle comprises a plurality of zwitterionic moieties.

In embodiments wherein the shell comprises a plurality ofsilane-functionalized zwitterionic moieties, the silane moieties and thezwitterionic moieties may be the same or different. For example, in oneembodiment, all the silane moieties may be the same and all thezwitterionic moieties may be the same. In another embodiment, the silanemoieties are the same but the zwitterionic moieties are different. Forexample, the shell may comprise two different silane-functionalizedzwitterionic moieties. The first one comprises a type 1 silane moietyand a type 1 zwitterionic moiety. The second one comprises a type 1silane moiety and a type 2 zwitterionic moiety, or a type 2 silanemoiety but a type 1 zwitterionic moiety, or a type 2 silane moiety and atype 2 zwitterionic moiety. In one or more embodiments, thesilane-functionalized zwitterionic moiety may comprise two or morezwitterionic moieties. In embodiments where the silane-functionalizedzwitterionic moiety comprises two or more zwitterionic moieties, thezwitterionic moieties may be the same or different.

In some embodiments, the silane-functionalized zwitterionic moietycomprises a positively charged moiety, a negatively charged moiety and afirst spacer group in between the positively charged moiety and thenegatively charged moiety. The positively charged moiety may originatefrom organic bases and the negatively charged moiety may originate fromorganic acids. FIG. 2 presents a list of exemplary organic acids andbases from which the negatively charged moiety and the positivelycharged moiety may originate.

In some embodiments, the positively charged moiety comprises protonatedprimary amines, protonated secondary amines, protonated tertiary alkylamines, protonated amidines, protonated guanidines, protonatedpyridines, protonated pyrimidines, protonated pyrazines, protonatedpurines, protonated imidazoles, protonated pyrroles, quaternary alkylamines, or combinations thereof.

In some embodiments, the negatively charged moiety comprisesdeprotonated carboxylic acids, deprotonated sulfonic acids, deprotonatedsulfinic acids, deprotonated phosphonic acids, deprotonated phosphoricacids, deprotonated phosphinic acids, or combinations thereof.

In one or more embodiments, the first spacer group comprises alkylgroups, aryl groups, substituted alkyl and aryl groups, heteroalkylgroups, heteroaryl groups, carboxy groups, ethers, amides, esters,carbamates, ureas, straight chain alkyl groups of 1 to 10 carbon atomsin length, or combinations thereof.

In some embodiments, a silicon atom of the silane-functionalizedzwitterionic moiety is connected to the positively or negatively chargedmoiety via a second spacer group. In some embodiments, the second spacergroup comprises alkyl groups, aryl groups, substituted alkyl and arylgroups, heteroalkyl groups, heteroaryl groups, carboxy groups, ethers,amides, esters, carbamates, ureas, straight chain alkyl groups of 1 to10 carbon atoms in length, or combinations thereof.

In some embodiments, the silane-functionalized zwitterionic moietycomprises the hydrolysis product of a precursor tri-alkoxy silane, suchas those illustrated in FIG. 3A-3D. In some embodiments, the precursortri-alkoxy silane comprisesN,N-dimethyl-3-sulfo-N-(3-(trimethoxysilyl)propyl)propan-1-aminium,3-(methyl(3-(trimethoxysilyl)propyl)amino) propane-1-sulfonic acid,3-(3-(trimethoxysilyl) propylamino)propane-1-sulfonic acid,2-(2-(trimethylsilyl)ethoxy(hydroxy)phosphoryloxy)-N,N,N-trimethylethanaminium,2-(2-(trimethoxysilyl)ethyl(hydroxy)phosphoryloxy)-N,N,N-trimethylethanaminium,N,N,N-trimethyl-3-(N-3-(trimethoxysilyl)propionylsulfamoyl)propan-1-aminium,N-((2H-tetrazol-5-yl)methyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,N-(2-carboxyethyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,3-(methyl(3-(trimethoxysilyl)propyl)amino)propanoic acid,3-(3-(trimethoxysilyl)propylamino) propanoic acid,N-(carboxymethyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,2-(methyl(3-(trimethoxysilyl)propyl)amino)acetic acid,2-(3-(trimethoxysilyl)propylamino) acetic acid,2-(4-(3-(trimethoxysilyl)propylcarbamoyl)piperazin-1-yl)acetic acid,3-(4-(3-(trimethoxysilyl)propylcarbamoyl)piperazin-1-yl)propanoic acid,2-(methyl(2-(3-(trimethoxysilyl)propylureido)ethyl)amino)acetic acid,2-(2-(3-(trimethoxysilyl) propylureido)ethyl)aminoacetic acid, orcombinations thereof.

Another aspect of the invention relates to a diagnostic agentcomposition. The diagnostic agent composition comprises a plurality ofthe nanoparticles 10 described previously. In one embodiment, thediagnostic agent composition further comprises a pharmaceuticallyacceptable carrier and optionally one or more excipients. In oneembodiment, the pharmaceutically acceptable carrier may be substantiallywater. Optional excipients may comprise one or more of salts,disintegrators, binders, fillers, or lubricants.

A small particle size may be advantageous in facilitating clearance fromkidneys and other organs, for example. In one embodiment, the pluralityof nanoparticles may have a median particle size up to about 50 nm. Inanother embodiment, the plurality of nanoparticles may have a medianparticle size up to about 10 nm. In another embodiment, the plurality ofnanoparticles may have a median particle size up to about 6 nm.

One aspect of the invention relates to methods for making a plurality ofnanoparticles. In general, one method comprises (a) providing a corehaving a core surface essentially free of silica, and (b) disposing ashell attached to the core surface, wherein the shell comprises asilane-functionalized zwitterionic moiety.

In one or more embodiments, the step of providing a core comprisesproviding a first precursor material, wherein the first precursormaterial comprises at least one transition metal. In one embodiment, thefirst precursor material reacts to generate the core comprising at leastone transition metal. In one embodiment, the first precursor materialdecomposes to generate the core. In another embodiment, the firstprecursor material hydrolyses to generate the core. In anotherembodiment, the first precursor material reacts to form the core.Nanoparticle synthesis methods are well known in the art and anysuitable method for making a nanoparticle core of an appropriatematerial may be suitable for use in this method.

In one or more embodiments, the step of disposing a shell comprisesproviding a second precursor material, such as a material comprising asilane moiety or a precursor to a silane moiety. The silane moiety mayreact with the core to form a shell comprising a silane moiety. In someembodiments, the precursor may undergo a hydrolysis reaction beforereacting with the core. In some embodiments, the silane moiety may befunctionalized with at least one zwitterionic moiety or at least oneprecursor to a zwitterionic moiety. In embodiments wherein the silanemoiety is functionalized with at least one zwitterionic moiety, theshell, thus formed, comprises a silane-functionalized zwitterionicmoiety. In embodiments wherein the silane moiety is functionalized witha precursor to a zwitterionic moiety, the shell, thus produced, may notbe zwitterionic in nature, but may subsequently react with anappropriate reagent to convert the precursor into a zwitterionic moiety.In one or more embodiments, the second precursor material comprises thesilane-functionalized zwitterionic moiety or precursor to asilane-functionalized zwitterionic moiety, such as one or more of theprecursor tri-alkoxy silanes described above.

It will be understood that the order and/or combination of steps may bevaried. Thus, according to some embodiments, steps (a) and (b) occur assequential steps so as to form the nanoparticle from the core and thesecond precursor material. By way of example and not limitation, in someembodiments, the first precursor material comprises at least onetransition metal; wherein the core comprises an oxide of the at leastone transition metal; and step (a) further comprises hydrolysis of thefirst precursor material. According to some embodiments, the firstprecursor material is an alkoxide or halide of the transition metal, andthe hydrolysis process includes combining the first precursor materialwith an acid and water in an alcoholic solvent. In some embodiments, thesilane may comprise polymerizable groups. The polymerization may proceedvia acid catalyzed condensation polymerization. In some otherembodiments, the silane moiety may be physically adsorbed on the core.In some embodiments, the silane moiety may be further functionalizedwith other polymers. The polymer may be water soluble and biocompatible.In one embodiment, the polymers include, but are not limited to,polyethylene glycol (PEG), polyethylene imine (PEI), polymethacrylate,polyvinylsulfate, polyvinylpyrrolidinone, or combinations thereof.

In some embodiments, the core comprises metal oxides. In one embodiment,the metal oxide core may be synthesized upon the hydrolysis of a metalalkoxide in the presence of an organic acid. In some embodiments, themetal alkoxide may be a tantalum alkoxide such as tantalum ethoxide, theorganic acid may be a carboxylic acid such as isobutyric acid, propionicacid or acetic acid and the hydrolysis reaction may be carried out inthe presence of an alcohol solvent such as 1-propanol or methanol.

In another embodiment, the core and the second precursor material may bebrought into contact to each other. In one embodiment, the secondprecursor material may comprise a silicon containing species such as anorganofunctional tri-alkoxysilane or mixture of organofunctionaltri-alkoxysilanes. At least one of the organofunctional tri-alkoxysilanes may contain at least one zwitterionic group or a precursor to azwitterionic group, such that each nanoparticle, on average, may containat least one zwitterionic moiety or precursor to a zwitterionic moiety.In one embodiment, each nanoparticle may contain on average, a pluralityof zwitterionic moieties or precursors to zwitterionic moieties. Inother embodiments, the core may be treated with a mixture containing atleast two silane moieties. In one embodiment, one silane moiety isfunctionalized with a zwitterionic moiety, or a precursor to azwitterionic moiety, and the second silane moiety may not befunctionalized with any zwitterionic moiety. The charged silane moietiesmay be added simultaneously or sequentially. In some embodiments, one ormore silane moieties functionalized with a zwitterionic moiety, or witha precursor to a zwitterionic moiety, may be added to the coresfunctionalized with non-zwitterionic silane moieties, eithersimultaneously or sequentially.

In one embodiment, a tantalum oxide core may be allowed to react with analkoxy silane that contains both, a quaternary nitrogen as well as asulfonate group or a carboxy group, for example, a sulfobetaine group ora betaine group. In one embodiment the tantalum oxide core may beallowed to react with (RO)₃Si(CH₂)_(x)NR′₂(CH₂)_(y)SO₃, where R is analkyl or aryl group, x is 1-10, y is 1-10, and R′ is H, an alkyl groupor an aryl group. In one embodiment, the R is an alkyl group, such asmethyl or ethyl, x is 3, y is between 2-5, and R′ is H or an alkyl groupsuch as methyl.

In one embodiment, sulfobetaine-functionalized silanes may besynthesised upon the ring opening reaction of alkyl sultones or amixture of alkyl sultones with amine substituted silanes. In anotherembodiment, alkyl lactones or mixtures of alkyl lactones may be used inplace of the alkyl sultones. In certain embodiments, the shell comprisesa mixture of sulfobetaine and betaine functional silanes. In anotherembodiment, the metal oxide core may react with a sulfobetaine orbetaine functional silane moiety, in which the sulfonate or carboxygroup may be chemically protected.

In another embodiment, the tantalum oxide core may be allowed to reactwith an amine-containing silane, such as an amino-functionaltrialkoxysilane, to form a tantalum oxide core functionalized with theamine-containing silane. In a second step, the core functionalized withthe silane may be isolated. In an alternative embodiment, the corefunctionalized with the silane may be used in-situ. The corefunctionalized with the silane may be allowed to react with an alkylsultone, an alkyl lactone, a haloalkylcarboxylic acid or ester, mixturesof alkyl sultones, mixtures of alkyl lactones, mixtures ofhaloalkylcarboxylic acids or esters, or mixtures of both alkyl sultonesand alkyl lactones to form a zwitterionic moiety. The amount of sultone,lactone or mixture of sultones and/or lactones may be sufficient toprovide, on average, at least one zwitterionic moiety per nanoparticle.Non-limiting examples of alkyl sultones include propane sultone andbutyl sultone. Non-limiting examples of lactones include propane lactoneand butyl lactone.

In one embodiment, the method further comprises fractionating theplurality of nanoparticles. The fractionating step may include filteringthe nanoparticles. In another embodiment, the method may furthercomprise purifying the plurality of nanoparticles. The purification stepmay include use of dialysis, tangential flow filtration, diafiltration,or combinations thereof. In another embodiment, the method furthercomprises isolation of the purified nanoparticles.

In combination with any of the above-described embodiments, someembodiments relate to a method for making a diagnostic agent compositionfor X-ray/computed tomography or MRI. The diagnostic agent compositioncomprises a plurality of nanoparticles. In some embodiments, the medianparticle size of the plurality of nanoparticles may not be more thanabout 10 nm, for example not more than about 7 nm, and in particularembodiments not more than about 6 nm. It will be understood thataccording to some embodiments, the particle size of the plurality ofnanoparticles may be selected so as to render the nanoparticlesubstantially clearable by a mammalian kidney, such as a human kidney,in particulate form.

In some embodiments, the present invention is directed to a method ofuse of the diagnostic agent composition comprising a plurality of thenanoparticles described herein. In some embodiments, the methodcomprises the in-vivo or in-vitro administration of the diagnostic agentcomposition to a subject, which in some instances may be a live subject,such as a mammal, and subsequent image generation of the subject with anX-ray/CT device. The nanoparticles, as described above, comprise a coreand a shell, wherein the shell comprises at least onesilane-functionalized zwitterionic moiety. In one embodiment, the corecomprises tantalum oxide. The nanoparticle may be introduced to thesubject by a variety of known methods. Non-limiting examples forintroducing the nanoparticle to the subject include intravenous,intra-arterial or oral administration, dermal application, or directinjection into muscle, skin, the peritoneal cavity or other tissues orbodily compartments.

In another embodiment, the method comprises administering the diagnosticagent composition to a subject, and imaging the subject with adiagnostic device. The diagnostic device employs an imaging method,examples of which include, but are not limited to, MRI, optical imaging,optical coherence tomography, X-ray, computed tomography, positronemission tomography, or combinations thereof. The diagnostic agentcomposition, as described above, comprises a plurality of thenanoparticles 10.

In one embodiment, the methods described above for use of the diagnosticcontrast agent further comprise monitoring delivery of the diagnosticagent composition to the subject with the diagnostic device, anddiagnosing the subject; in this method data may be compiled and analyzedgenerally in keeping with common operation of medical diagnostic imagingequipment. The diagnostic agent composition may be an X-ray or CTcontrast agent, for example, such as a composition comprising a tantalumoxide core. The diagnosing agent composition may provide for a CT signalin a range from about 100 Hounsfield to about 5000 Hounsfield units. Inanother example, the diagnostic agent composition may be a MRI contrastagent, such as an agent comprising a superparamagnetic iron oxide core.

One embodiment of the invention provides a method for determination ofthe extent to which the nanoparticles 10 described herein, such asnanoparticles having tantalum oxide or iron oxide cores, are distributedwithin a subject. The subject may be a mammal or a biological materialcomprising a tissue sample or a cell. The method may be an in-vivo orin-vitro method. The nanoparticle may be introduced to the subject by avariety of known methods. Non-limiting examples for introducing thenanoparticle to the subject include any of the known methods describedabove. In one embodiment, the method comprises (a) introducing thenanoparticles into the subject, and (b) determining the distribution ofthe nanoparticles in the subject. Distribution within a subject may bedetermined using a diagnostic imaging technique such as those mentionedpreviously. Alternatively, the distribution of the nanoparticle in thebiological material may be determined by elemental analysis. In oneembodiment, Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) may beused to determine the concentration of the nanoparticle in thebiological material.

The following examples are included to demonstrate particularembodiments of the present invention. It should be appreciated by thoseof skill in the art that the methods disclosed in the examples thatfollow merely represent exemplary embodiments of the present invention.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments described and still obtain a like or similar result withoutdeparting from the spirit and scope of the present invention.

EXAMPLES

Practice of the invention will be still more fully understood from thefollowing examples, which are presented herein for illustration only andshould not be construed as limiting the invention in any way.

The abbreviations used in the examples section are expanded as follows:“mg”: milligrams; “mL”: milliliters; “mg/mL”: milligrams per milliliter;“mmol”: millimoles; “μL” and μLs: microliters “LC”: LiquidChromatography; “DLS”: Dynamic Light Scattering; “DI”: Deionized water,“ICP”: Inductively Coupled Plasma.

Unless otherwise noted, all reagent-grade chemicals were used asreceived, and Millipore water was used in the preparation of all aqueoussolutions.

Synthesis of Tantalum Oxide-Based Nanoparticles

Step-1: Synthesis ofN,N-dimethyl-3-sulfo-N-(3-(trimethoxysilyl)propyl)propan-1-aminium

Toluene (anhydrous, 250 mL), N,N-dimethylaminotrimethoxysilane (25 g,121 mmol) and 1,3-propane sultone (13.4 g, 110 mmol) were added to a 500mL round bottom flask containing a stir bar. The mixture was stirred atroom temperature for 4 days. The mixture was then filtered to isolatethe precipitated product, which was subsequently washed with freshanhydrous toluene (2×60 mL). The yield of white powder after dryingunder vacuum was 23.6 g.

Step-2: Reaction ofN,N-dimethyl-3-sulfo-N-(3-(trimethoxysilyl)propyl)propan-1-aminium withTantalum oxide based core

Method-1: 1-propanol as Solvent

A 250 mL three necked round bottomed flask containing a stir bar wascharged with 1-propanol (73 mL), followed by addition of isobutyric acid(1.16 mL, 12.51 mmol, 1.27 eq with respect to Ta) and DI water (1.08 mL,59.95 mmol, 6.09 eq with respect to Ta) to form a reaction mixture.Nitrogen was bubbled through the reaction mixture for 20 minutesfollowed by dropwise addition of tantalum ethoxide (Ta(OEt)₅) (2.55 mL,4 g, 9.84 mmol) to the reaction mixture at room temperature withstirring over 15 minutes. During the addition of Ta(OEt)₅, the nitrogenwas continued to bubble through the reaction mixture. The abovementioned reaction mixture was allowed to stir at room temperature undernitrogen for 16 hours after the Ta(OEt)₅ addition was complete.

The reaction mixture was stirred at room temperature for 16 hours andthen an aliquot (1.5 mL) was taken out from the reaction mixture,filtered through a 20 nm filtration membrane, and the particle size wasmeasured (as the hydrodynamic radius) in water by DLS immediately afterthe filtration step. The average particle size was measured to beapproximately 3.6 nm.N,N-dimethyl-3-sulfo-N-(3-(trimethoxysilyl)propyl)propan-1-aminium (4.03g, 12.23 mmol, 1.24 eq with respect to Ta) was dissolved in 50 mL of DIwater. This solution was added to the above mentioned reaction mixturedropwise over a few minutes. The colorless, homogeneous reaction mixturewas changed immediately into a cloudy white solution and finally becamea milky solution by the end of the addition of the silane-functionalizedzwitterionic moiety. After the addition was complete a condenser wasattached to the flask, and the reaction mixture was kept under anitrogen blanket. The flask was placed in an oil bath preheated to 75°C. and the reaction mixture was stirred for 6 hours. The reactionmixture became clearer. After 6 hours, the reaction mixture was cooledto room temperature under a blanket of air. The heterogeneous reactionmixture was neutralized to pH 6-7 using 1(M) NH₄OH. The reaction mixturewas transferred into a second round bottom flask under a blanket of air.During the transfer of the reaction mixture to the second flask, anamount of white material remained in the flask, and did not gettransferred to the second flask (crude product A). This crude product Awas dried under a flow of nitrogen overnight. Meanwhile, the solution ofthe second flask was evaporated using a rotary evaporator at 50° C. Thedry white residue obtained after the evaporation of the solution, (crudeproduct B) was allowed to stand under a nitrogen flow over night.

The crude product A was dried overnight. This solid was completelydissolved in DI water. Crude product B was also completely dissolved inDI water, and the two solutions (crude product A & crude product B) werecombined (total volume was 60 mL). The aqueous solution was filteredsequentially through 450 nm, 200 nm and 100 nm filtration membranes andfinally through a 20 nm filtration membrane. The solution was then firstdialyzed at pH 7.0 using sodium phosphate buffer (10K molecular weightcut-off snakeskin regenerated cellulose tubing), and then three times inDI water.

Finally, the nanoparticle was isolated by lyophilization. Yield of whitepowder=1.748 g (38% yield based on Ta). Zeta potential: (−)8.18 mV.Elemental analysis: 38.3±0.3% Ta, 4.8±0.1% Si. The average particle sizewas measured to be 8.9 nm by DLS. Purity of the nanoparticle wasmeasured by Liquid Chromatography (LC)/Inductively Coupled Plasma (ICP).

Method-2: Trifluoroethanol as Solvent

A 100 mL three necked round bottom flask containing a stir bar wascharged with trifluoroethanol (42 mL). While the solvent was spargedwith nitrogen, isobutyric acid (0.53 mL, 5.7 mmol) followed by water(0.13 mL, 7.4 mmol) were added using a syringe. The solution was allowedto stir for an additional 15 min with continuous nitrogen bubbling.Tantalum ethoxide (Ta(OEt)₅) (2 g, 4.9 mmol) was added dropwise using asyringe. The slightly hazy solution was allowed to stir at roomtemperature under nitrogen for 17 hours.N,N-dimethyl-3-sulfo-N-(3-(trimethoxysilyl)propyl)propan-1-aminium(example 1, 3.2 g, 9.8 mmol) was dissolved in water (15 mL). Thishomogeneous, colorless solution was added to the tantalum containingreaction mixture dropwise but quickly under air with stirring. The flaskwas fitted with a condenser and then placed in an oil bath preheated to78° C. After stirring at this temperature for 6 hours, the colorless,homogeneous reaction mixture was cooled to room temperature.Trifluoroethanol was substantially removed in a rotary evaporator afteradding water (20 mL). The aqueous solution was neutralized usingconcentrated ammonium hydroxide and then filtered successively through200 nm, 100 nm and then 20 nm filters. The solution was then dialyzedusing 3500 MW cut-off regenerated cellulose snake skin dialysis tubing 4times. The first dialysis was performed in 50:50 DI water to pH 7.0phosphate buffer. Subsequent dialyses were performed in DI water. Thepurified nanoparticle product was not isolated from water. A percentsolids test on an aliquot was used to determine that the yield of coatednanoparticles was 1.55 g. The average particle size was determined bydynamic light scattering to be 1.6 nm.

Synthesis of Tantalum Oxide-Based Nanoparticle

Step-1: Synthesis of Ethyl2(4(3(trimethoxysilyl)propylcarbamoyl)piperazin-1-yl)acetate

(3-isocyanatopropyl)trimethoxysilane (4.106 g) was added to a solutionof ethylacetoxypiperazine (3.789 g) in methylene chloride (20 mL). Thesolution was stirred for 16 hours, and then the solvent was removedunder reduced pressure, yielding 8.37 g of material that was usedwithout further purification.

Step-2: Reaction of Ethyl2-(4-(3-(trimethoxysilyl)propylcarbamoyl)piperazin-1-yl)acetate withTantalum Oxide-Based Core

A 500 mL round-bottom flask was charged with n-propanol (99 mL),isobutyric acid (1.4 mL), and water (1.2 mL). The solution was stirredfor 5 min., then Ta(OEt)₅ (5.37 g) was added dropwise to the solution.The solution was stirred at room temperature under nitrogen for 18hours. A total of 60 mL of this solution was then added to ethyl2-(4-(3-(trimethoxysilyl)propylcarbamoyl)piperazin-1-yl)acetate (6.37g), and the solution was stirred under nitrogen for 2 hours at 100° C.The mixture was then cooled to room temperature, water (20 mL) wasadded, and the mixture was stirred for 18 hours at room temperature. Atotal of 75 mL of 0.33 N aqueous hydrochloric acid was then added, andthe solution was heated to 60° C. for 6 hours. The mixture was thencooled to room temperature, 250 mL of 28% aqueous ammonia was added, andthe mixture was stirred for 5 days. The ammonia and propanol wereremoved under reduced pressure, then the material was poured into 3,000MW cut-off regenerated cellulose dialysis tubing, and dialyzed againstdistilled water for 48 hours, changing the dialysis buffer every 12hours. The solution was then filtered through 30,000 MW cut-offcentrifuge filters, yielding particles with an average size of 4.5 nm,as measured by DLS.

Synthesis of Iron Oxide-Based Nanoparticle

Synthesis of Superparamagnetic Iron Oxide Nanoparticles

A 100 mL three-necked round bottom flask was charged with 706 mg ofFe(acac)₃ and 20 mL of anhydrous benzyl alcohol. The solution wassparged with nitrogen and then heated to 165° C. for 2 hours under anitrogen atmosphere. The solution was then cooled to, and stored, atroom temperature.

Reaction of Ethyl2-(4-(3-(trimethoxysilyl)propylcarbamoyl)piperazin-1-yl)acetate withSuperparamagnetic Iron Oxide

A 10 mL aliquot of superparamagnetic iron oxide nanoparticles in benzylalcohol (5.58 mg Fe/mL) was diluted with 50 mL of tetrahydrofuran. 2.00g of ethyl2-(4-(3-(trimethoxysilyl)propylcarbamoyl)piperazin-1-yl)acetate wasadded, and the mixture was heated to 60° C. with stirring for 2 hours,followed by cooling to room temperature. 50 mL of 1.0 M aqueouspotassium carbonate was added after which the flask was then sealed andheated with stirring to 60° C. for 18 hours. The mixture was then cooledand centrifuged, and the aqueous layer was poured into 10,000 MW cut-offregenerated cellulose dialysis tubing and dialyzed vs 4 liters of 10 mMsodium citrate for 48 hours, changing the dialysis buffer every 12hours. The final volume was 94 mL, with a total of 0.416 mg iron per mLof solution. The material had an average particle size of 8.4 nm in 150mM aqueous sodium chloride as measured by dynamic light scattering.

Reaction ofN,N-dimethyl-3-sulfo-N-(3-(trimethoxysilyl)propyl)propan-1-aminium withSuperparamagnetic Iron Oxide

A 16.75 mL aliquot of superparamagnetic iron oxide nanoparticles inbenzyl alcohol (5.58 mg Fe/mL) was added to tetrahydrofuran for a totalvolume of 94.5 mL. This solution was then added to a pressure flask,along with 3.1 g ofN,N-dimethyl-3-sulfo-N-(3-(trimethoxysilyl)propyl)propan-1-aminium, andthe mixture was heated to 50° C. with stirring for 2 hours. Aftercooling to room temperature, a total of 31 mL of isopropanol and 76 mLof concentrated aqueous ammonium hydroxide (28% NH₃ in water) wereadded; the flask was then sealed and heated to 50° C. with stirring for18 hours. The mixture was cooled and washed with hexanes (100 mL×3). Theaqueous layer was poured into 10,000 MW cut-off regenerated cellulosedialysis tubing, and dialyzed vs 4 liters of 10 mM sodium citrate for 18hours. The final solution had a total of 0.67 mg iron per mL ofsolution. The material had a particle size of 9.2 nm.

Determination of the Particle Size and Stability of the Nanoparticles inWater

Nanoparticles from method 1 (36.2 mg) were dissolved in 2 mL of DIwater. The solution was filtered through a 20 nm filtration membrane.The average particle size was measured as a hydrodynamic radius bydynamic light scattering (DLS), immediately after the filtration step.The sample was stored for 15 days at 37° C., with periodic monitoring byDLS. The results are shown in Table 1.

TABLE 1 Time (t) Average particle size*  0 10.1 nm  5 days 12.8 nm 15days 12.2 nm *Average particle size was measured at 37° C., using DLS.

Nanoparticle Biodistribution Studies

In-vivo studies were carried out with male Lewis rats with a size rangebetween 150 and 500 grams body weight. Rats were housed in standardhousing with food and water ad libitum and a 12 hour day-night lightingcycle. All animals used for biodistribution were otherwise untreated,normal subjects.

Nanoparticles were administered as a filter-sterilized solution ineither water or saline. Administration was performed under isofluraneanesthesia (4% induction, 2% maintenance) via a 26 G catheter insertedinto the lateral tail vein. Injection volumes were determined based onthe concentration of the nanoparticles in the injectate and the size ofthe rat, but were generally less than 10% of rodent blood volume. Thetarget dose was 100 mg of core metal (e.g., tantalum) per kg of bodyweight. Once injected, animals were removed from anesthesia and, after aperiod of observation for adverse effects, returned to normal housing.At a later period of as short as a few minutes to as long as 6 months,the rats were euthanized, and organs of interest were harvested,weighed, and analyzed for their total metal (e.g., tantalum) content byICP analysis. Along with the organs, a sample of the injected materialwas submitted to determine the exact concentration of injectate. Thesecombined data determined the percentage of the injected dose (“% ID”)remained in a tissue of interest. These data were reported either as %ID/organ, or % ID/gram of tissue. Experiments were generally performedwith four duplicate rats at each time-point, allowing for thedetermination of experimental error (±standard deviation).

TABLE 2 Kidney Liver Spleen Coating (% ID/organ) (% ID/organ) (%ID/organ) Diethylphosphatoethyl-  4.2 ± 0.43 2.57 ± 0.64 0.16 ± 0.05triethoxysilane (PHS) N,N-dimethyl-3-sulfo-N-(3- 0.29 ± 0.05 0.24 ± 0.02ND (trimethoxysilyl)- propyl)propan-1-aminium (SZWIS)N-(2-carboxyethyl)-N,N- 0.70 ± 0.47 0.33 ± 0.03 0.04 ± 0.01dimethyl-3-(trimethoxysilyl)- propan-1-aminium (CZWIS)

Table-2 describes the biodistribution of fractionated nanoparticles withnon-zwitterionic (PHS) and zwitterionic coatings (SZWIS and CZWIS) inmajor clearing organs at 1 week following IV injection. “ND” stands for“not detected”.

The amount of tantalum retained per organ is represented in the Table-2as the fraction of the injected dose. Comparably sized non-zwitterioniccoated nanoparticles are retained at much higher levels (almost oneorder of magnitude) than either of the zwitterionic coatings tested.

While only certain features of the invention have been illustrated anddescribed herein, many modifications and changes will occur to thoseskilled in the art. It is, therefore, to be understood that the appendedclaims are intended to cover all such modifications and changes as fallwithin the true spirit of the invention.

The invention claimed is:
 1. A method comprising: administering adiagnostic agent composition to a subject, wherein the diagnostic agentcomposition comprises a plurality of nanoparticles, wherein thenanoparticle comprises a core, having a core surface essentially free ofsilica; and a shell attached to the core surface; wherein the shellcomprises at least one silane-functionalized zwitterionic moiety,wherein the silane functionalized zwitterionic moiety comprises a silanemoiety and a zwitterionic moiety, and wherein the silane moiety iscovalently bonded to the zwitterionic moiety via a spacer comprisingless than 10 carbon atoms; and wherein the silane-functionalizedzwitterionic moieties are bound to the core at the core surface withoutany intervening silica layer; wherein the silane functionalizedzwitterionic moiety comprises a hydrolysis product of a precursortri-alkoxy silane wherein the precursor tri-alkoxy silane is selectedfrom a group consisting ofN,N-dimethyl-3-sulfo-N-(3-(trimethoxysilyl)propyl)propan-1-aminium,3-(methyl(3-(trimethoxysilyl)propyl)amino) propane-l-sulfonic acid,3-(3-(trimethoxysilyl) 5 propylamino)propane-1-sulfonic acid,2-(2-(trimethylsilyl)ethoxy(hydroxy)phosphoryloxy)-N,N,N-trimethylethanaminium,2-(2-(trimethoxysilyl)ethyl(hydroxy)phosphoryloxy)-N,N,N-trimethylethanaminium,N,N,N-trimethyl-3-(N-3-(trimethoxysilyl)propionylsulfamoyl)propan-1-aminium,N-((2H-tetrazol-5-yl)methyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,N-10(2-carboxyethyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,3-(methyl(3-(trimethoxysilyl)propyl)amino)propanoic acid,3-(3-(trimethoxysilyl)propylamino)propanoic acid,N-(carboxymethyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,2-(methyl(3-(trimethoxysilyl)propyl)amino)acetic acid,2-(3-(trimethoxysilyl)propylamino)acetic acid,2-(4-(3-(trimethoxysilyl)propylcarbamoyl)15 piperazin-1-yl)acetic acid,3-(4-(3-(trimethoxysilyl)propylcarbamoyl)piperazin-1-yl) propanoic acid,2-(methyl(2-(3-(trimethoxysilyl) propylureido)ethyl)amino)acetic acid,2-(2-(3-(trimethoxysilyl)propylureido)ethyl)amino acetic acid, andcombinations thereof and imaging the subject with a diagnostic device.2. The method of claim 1, further comprises monitoring delivery of thediagnostic agent composition to the subject with the diagnostic device;and diagnosing the subject.
 3. The method of claim 1, wherein thediagnostic agent composition is administered to a subject in-vitro or invivo.
 4. The method of claim 1, wherein the diagnostic device employs animaging method selected from the group consisting of MRI, opticalimaging, optical coherence tomography, X ray, computed tomography,positron emission tomography, or combinations thereof.
 5. The method ofclaim 1, wherein the core comprises a transition metal.
 6. The method ofclaim 1, wherein the core comprises tungsten, tantalum, hafnium,zirconium, molybdenum, silver, zinc, or combinations thereof.
 7. Themethod of claim 1, wherein the core comprises tantalum oxide.
 8. Themethod of claim 1, wherein the core comprises a superparamagneticmaterial.
 9. The method of claim 1, wherein the core comprisessuperparamagnetic iron oxide.
 10. The method of claim 1, wherein the thezwitterionic moiety comprises protonated primary amines, protonatedsecondary amines, protonated tertiary alkyl amines, protonated amidines,protonated guanidines, protonated pyridines, protonated pyrimidines,protonated pyrazines, protonated purines, protonated imidazoles,protonated pyrroles, quaternary alkyl amines, or combinations thereof.11. The method of claim 1, wherein the the zwitterionic moiety comprisesdeprotonated carboxylic acids, deprotonated sulfonic acids, deprotonatedsulfinic acids, deprotonated phosphonic acids, deprotonated phosphoricacids, deprotonated phosphinic acids, or combinations thereof.
 12. Themethod of claim 1, wherein the plurality of nanoparticles has a medianparticle size up to about 50 nm.
 13. The method of claim 1, wherein thespacer is selected from a group consisting of alkyl group, aryl group,substituted alkyl and aryl group, heteroalkyl group, heteroaryl group,carboxy group, ether, amide, ester, carbamate, urea, straight chainalkyl group of 1 to 10 carbon atoms in length and combination thereof.14. The method of claim 1, wherein the silane functionalizedzwitterionic moiety having structure I


15. The method of claim 1, wherein the silane functionalizedzwitterionic moiety having structure II


16. The method of claim 1, wherein the nanoparticle further comprises atargeting ligand.
 17. A method comprising: administering a diagnosticagent composition to a subject, wherein the diagnostic agent compositioncomprises a plurality of nanoparticles, wherein the nanoparticlecomprises a core having a core surface; wherein the core comprisestantalum oxide, and a shell attached to the core surface; wherein theshell comprises at least one silane-functionalized zwitterionic moiety,and imaging the subject with an X-ray device.
 18. A method comprising:administering a diagnostic agent composition to a subject, wherein thediagnostic agent composition comprises a plurality of nanoparticles,wherein the nanoparticle comprises a core, having a core surfaceessentially free of silica; and a shell attached to the core surface;wherein the shell comprises at least one silane-functionalizedzwitterionic moiety, wherein the silane functionalized zwitterionicmoiety comprises a silane moiety and a zwitterionic moiety, and whereinthe silane moiety is covalently bonded to the zwitterionic moiety; andwherein the silane-functionalized zwitterionic moieties are bound to thecore at the core surface without any intervening silica layer; whereinthe silane functionalized zwitterionic moiety comprises a hydrolysisproduct of a precursor tri-alkoxy silane wherein the precursortri-alkoxy silane is selected from a group consisting ofN,N-dimethyl-3-sulfo-N-(3-(trimethoxysilyl)propyl)propan-1-aminium,3-(methyl(3-(trimethoxysilyl)propyl)amino) propane-l-sulfonic acid,3-(3-(trimethoxysilyl) 5 propylamino)propane-1-sulfonic acid,2-(2-(trimethylsilyl)ethoxy(hydroxy)phosphoryloxy)-N,N,N-trimethylethanaminium,2-(2-(trimethoxysilyl)ethyl(hydroxy)phosphoryloxy)-N,N,N-trimethylethanaminium,N,N,N-trimethyl-3-(N-3-(trimethoxysilyl)propionylsulfamoyl)propan-1-aminium,N-((2H-tetrazol-5-yl)methyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,N-10(2-carboxyethyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,3-(methyl(3-(trimethoxysilyl)propyl)amino)propanoic acid,3-(3-(trimethoxysilyl)propylamino)propanoic acid,N-(carboxymethyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,2-(methyl(3-(trimethoxysilyl)propyl)amino)acetic acid,2-(3-(trimethoxysilyl)propylamino)acetic acid,2-(4-(3-(trimethoxysilyl)propylcarbamoyl)15 piperazin-1-yl)acetic acid,3-(4-(3-(trimethoxysilyl)propylcarbamoyl)piperazin-1-yl) propanoic acid,2-(methyl(2-(3-(trimethoxysilyl) propylureido)ethyl)amino)acetic acid,2-(2-(3-(trimethoxysilyl)propylureido)ethyl)amino acetic acid, andcombinations thereof and wherein the nanoparticle does not contain atargeting ligand; and imaging the subject with a diagnostic device. 19.A method comprising: administering a diagnostic agent composition to asubject, wherein the diagnostic agent composition comprises a pluralityof nanoparticles, wherein the nanoparticle comprises a core, having acore surface essentially free of silica; and a shell attached to thecore surface; wherein the shell comprises at least onesilane-functionalized zwitterionic moiety, wherein the silanefunctionalized zwitterionic moiety comprises a silane moiety and azwitterionic moiety, and wherein the silane moiety is covalently bondedto the zwitterionic moiety; and wherein the silane-functionalizedzwitterionic moieties are bound to the core at the core surface withoutany intervening silica layer; wherein the silane functionalizedzwitterionic moiety comprises a hydrolysis product of a precursortri-alkoxy silane wherein the precursor tri-alkoxy silane is selectedfrom a group consisting ofN,N-dimethyl-3-sulfo-N-(3-(trimethoxysilyl)propyl)propan-1-aminium,3-(methyl(3-(trimethoxysilyl)propyl)amino) propane-l-sulfonic acid,3-(3-(trimethoxysilyl) 5 propylamino)propane-1-sulfonic acid,2-(2-(trimethylsilyl)ethoxy(hydroxy)phosphoryloxy)-N,N,N-trimethylethanaminium,2-(2-(trimethoxysilyl)ethyl(hydroxy)phosphoryloxy)-N,N,N-trimethylethanaminium,N,N,N-trimethyl-3-(N-3-(trimethoxysilyl)propionylsulfamoyl)propan-1-aminium,N-((2H-tetrazol-5-yl)methyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,N-10(2-carboxyethyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,3-(methyl(3-(trimethoxysilyl)propyl)amino)propanoic acid,3-(3-(trimethoxysilyl)propylamino)propanoic acid,N-(carboxymethyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium,2-(methyl(3-(trimethoxysilyl)propyl)amino)acetic acid,2-(3-(trimethoxysilyl)propylamino)acetic acid,2-(4-(3-(trimethoxysilyl)propylcarbamoyl)15 piperazin-1-yl)acetic acid,3-(4-(3-(trimethoxysilyl)propylcarbamoyl)piperazin-1-yl) propanoic acid,2-(methyl(2-(3-(trimethoxysilyl) propylureido)ethyl)amino)acetic acid,2-(2-(3-(trimethoxysilyl)propylureido)ethyl)amino acetic acid, andcombinations thereof and a targeting ligand, wherein the zwitterionicmoiety and the targeting ligand are not the same; and imaging thesubject with a diagnostic device.